This Project focuses on Protein Tyrosine Phosphatase (PTP)mu regulation of the endothelial cell (EC)-EC adherens junction or the zonula adherens (ZA), ZA linkage to the actin cytoskeleton, and actin organization. The project leader will apply his background in ZA-actin cytoskeleton biology together with Dr. A. Passanitrs expertise in angiogenesis to address the mechanisims through which PTPImu and the ZA-actin contractile apparatus supports the dynamic EC-EC interactions that are required during tyrosine phosphorylation-driven angiogenesis. Several PTPs that associate with and/or dephosphorylate components of the ZA multiprotein complex appear to regulate the state of ZA protein tyrosine phosphorylation and assembly. One such PTP, PTPmu, is highly expressed in lung tissue, almost exclusively expressed in EC, binds to the cytoplasmic domain of vascular endothelial (VE)-cadherin and/or to one or more of the catenins, and through its own ectodomain participates in homophilic adhesion. We will use human lung microvascular EC as well as an immortalized human microvascular EC line together with a retrovirus gene transfer system to stably overexpress wild-type PTPmu, catalytically-inactive PTPmu mutants, antisense PTPmu and selected PTPmu domains. Tube formation, coimmunoprecipitation, and GST-PTPmu substrate-trapping mutant binding assays, phosphotyrosine immunoblotting, recombinant protein expression, fluorescence microscopy, and actin pool measurements will be used to address four Specific Aims. SA# 1 will determine whether PTPmu catalytic function regulates in vitro angiogenesis. SA# 2 will identify PTPmu substrates in human microvascular EC that are established mediators of angiogenesis. SA# 3 will determine whether homophilic interactions between PTPmu ectodomains regulate PTPmu catalytic activity and angiogenesis. Finally, SA# 4 will determine whether PTPmu catalytic and/or ectodomain functions regulate the ZA-actin cytoskeletal linkage and/or actin organization. These studies will test whether PTPmu serves a counter-regulatory role in the tightly orchestrated formation and subsequent stability of new vessels during tyrosine phosphorylation dependent, cytoskeleton-driven angiogenesis.